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. 2019 Nov 14;9(1):16835.
doi: 10.1038/s41598-019-53383-z.

Time-lapse image analysis for whole colony growth curves and daily distribution of the cell number per colony during the expansion of mesenchymal stem cells

Mitsuru Mizuno  1 , Hisako Katano  1 , Yuri Shimozaki  2 , Sho Sanami  2 , Nobutake Ozeki  1 , Hideyuki Koga  3 , Ichiro Sekiya  4
Affiliations

Time-lapse image analysis for whole colony growth curves and daily distribution of the cell number per colony during the expansion of mesenchymal stem cells

Mitsuru Mizuno et al. Sci Rep. .

Abstract

Mesenchymal stem cells from the synovium (synovial MSCs) are attractive for cartilage and meniscus regeneration therapy. We developed a software program that can distinguish individual colonies and automatically count the cell number per colony using time-lapse images. In this study, we investigated the usefulness of the software and analyzed colony formation in cultured synovial MSCs. Time-lapse image data were obtained for 14-day-expanded human synovial MSCs. The cell number per colony (for 145 colonies) was automatically counted from phase-contrast and nuclear-stained images. Colony growth curves from day 1 to day 14 (for 140 colonies) were classified using cluster analysis. Correlation analysis of the distribution of the cell number per colony at 14 days versus that number at 1-14 days revealed a correlation at 7 and 14 days. We obtained accurate cell number counts from phase-contrast images. Individual colony growth curves were classified into three main groups and subgroups. Our image analysis software has the potential to improve the evaluation of cell proliferation and to facilitate successful clinical applications using MSCs.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Multi-colored images of synovial MSC colony formation. Synovial MSCs were plated at 20 cells/cm2 and cultured. When single cells had proliferated to yield four cells, the colonies were colored and counted using our software. White circles show single cell-derived colonies, and yellow circles show a single colony consisting of nine single cell-derived colonies.
Figure 2
Figure 2
The cell number per colony evaluated from images converted to black and white (BW) and DAPI images. (A) Black and white images and DAPI signal images of synovial MSCs. Cells were plated at 20 cells/cm2 and cultured. Images were converted to BW using our software, and the DAPI signal appears as blue. (B) Correlation between the cell number per colony from BW converted images and DAPI signal images analyzed by Spearman’s correlation coefficient.
Figure 3
Figure 3
Individual growth curves of the cell number per colony in MSC colonies. Synovial MSCs were plated at 20 cells/cm2 and cultured for 14 days. In total, 140 colonies were analyzed using our software.
Figure 4
Figure 4
Classification of MSC colony growth curves by the cell number per colony. (A) Cluster analysis based on the growth curve of the cell number per colony, which was classified into large 1,2, medium 1–4, and small groups. (B) Individual growth curve of the cell number per colony in each group.
Figure 5
Figure 5
Analysis of cell colonies in the large 1,2, medium 1–4, and small groups. Synovial MSCs were plated at 20 cells/cm2 and cultured for 14 days. (A) Representative colony images. (B) Representative black and white converted images in each group. (C) Quantitative analysis of the cell long axis in each colony. (D) Quantitative analysis of the distance between cells in each colony. *P < 0.05 by Kruskal Wallis test and Dunn’s multiple comparisons test.
Figure 6
Figure 6
Correlation analysis of the cell number per colony on day 14 and days 1–14. Synovial MSCs were plated at 20 cells/cm2 and cultured for 14 days. The cell number per colony was counted daily using our software.
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